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Orientador: Fernanda Paula Collares Queiroz / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-18T11:02:37Z (GMT). No. of bitstreams: 1 CastroTerezan_VaninaHelende_D.pdf: 2092752 bytes, checksum: aa76b9dfefe523915640c52fb45ac5e3 (MD5) Previous issue date: 2011 / Resumo: Neste trabalho, foram desenvolvidos, pelo processo de extrusão termoplástica, salgadinhos expandidos à base de farinha de milho enriquecidos com a incorporação de farinha de quinoa, visando o aumento do valor nutricional. O milho é muito utilizado na fabricação de salgadinhos pelo fato de expandir com facilidade. A quinoa (Chenopodium quinoa) é um pseudocereal de importante potencial agronômico e alto valor nutricional, apresentando elevado teor de proteína, com expressivos níveis do aminoácido lisina. As matérias-primas foram caracterizadas quanto às propriedades físicas, químicas e nutricionais. Para verificar quais parâmetros influenciariam as propriedades dos salgadinhos expandidos, primeiramente foi realizado um delineamento fatorial fracionado 25-1, cujas variáveis independentes foram: (i) umidade inicial da amostra; (ii) teor de farinha de quinoa; (iii) temperatura da 4ª zona do extrusor; (iv) temperatura da 5ª zona do extrusor e (v) velocidade da rosca. As variáveis dependentes avaliadas foram: (i) índice de expansão, (ii) dureza e (iii) lisina biodisponível. As variáveis que influenciaram significativamente pelo menos uma das respostas, considerando p<0,20, foram: (i) umidade inicial da amostra, (ii) teor de farinha de quinoa e (iii) temperatura da 5ª zona do extrusor. Na sequência, foi executado um Delineamento Composto Central Rotacional (DCCR), com essas três variáveis independentes, alterando as faixas de estudos em função do impacto que as mesmas apresentaram sobre as respostas. Não foi possível gerar modelos matemáticos válidos que descrevessem a tendência da dureza e da lisina biodisponível nas faixas de variação estudadas (umidade de 15 a 20%, teor de farinha de quinoa entre 15 e 50% e temperatura da 5ª zona do extrusor de 110 a 160ºC). O aumento destas três variáveis independentes promoveu a redução do índice de expansão. Pela análise de superfície de resposta, observou-se que as condições para a produção de salgadinhos extrudados com alta expansão foram: umidade inicial da amostra em 17,5%, adição de 22% de farinha de quinoa e temperatura de 110ºC na 5ª zona do extrusor. Nestas condições, foram produzidos salgadinhos expandidos apenas de milho (padrão) e salgadinhos com incorporação de 22 e 43% de quinoa, que foram caracterizados, aromatizados e submetidos a uma avaliação sensorial de aceitação e intenção de compra. Os salgadinhos expandidos de milho e quinoa apresentaram dureza e diâmetros similares aos disponíveis no mercado, indicando que as condições do processo de extrusão foram adequadamente escolhidas. Do ponto de vista nutricional, os salgadinhos com quinoa apresentaram maior teor de proteína, aminoácidos essenciais (treonina, cisteína, isoleucina, lisina e triptofano) e lisina biodisponível, em relação ao salgadinho apenas de milho, mostrando a viabilidade da adição de farinha de quinoa em produtos à base de milho para aumentar o seu valor nutricional. O salgadinho expandido com 22% farinha de quinoa apresentou boa aceitação e alta intenção de compra, enquanto o salgadinho com 43% não foi bem aceito, por apresentar menor expansão, escurecimento e sabor residual / Abstract: In this work, expanded snacks made from corn flour enriched by incorporating quinoa flour with the aim of increasing the nutritional value, were developed and processed by thermoplastic extrusion. Corn is widely used in snack production since it expands easily. Quinoa (Chenopodium quinoa) is a pseudocereal with important agronomic potential and a high nutritional value, containing an elevated protein content and expressive amount of the amino acid lysine. The raw materials were characterized with respect to their physical, chemical and nutritional properties. Initially a 25-1 Fractional Factorial Design was used to determine which parameters influenced the properties of the expanded snacks, the independent variables being: (i) initial moisture content; (ii) amount of quinoa flour; (iii) temperature of the 4th extruder zone; (iv) temperature of the 5th extruder zone and (v) screw speed. The dependent variables evaluated were: (i) expansion ratio; (ii) hardness and (iii) available lysine. The independent variables that significantly influenced at least one of the responses, considering p<0.20, were: (i) initial moisture content; (ii) amount of quinoa flour; and (iii) temperature of the 5th extruder zone. In sequence, a Central Composite Rotatable Design (CCRD) was carried out with the above three independent variables, altering the range of the study considering the impact they had on the responses. It was not possible to obtain valid mathematical models to describe the trends with respect to hardness and available lysine in the range of variation studied (moisture content from 15 to 20%, quinoa flour content from 15 to 50% and temperature in the 5th extruder zone from 110 to 160ºC). Increases in these three independent variables resulted in a reduction in the expansion ratio. From the analysis of the response surface, it was observed that the conditions required to produce highly expanded extruded snacks were: initial moisture content of 17.5%, addition of 22% of quinoa flour and a temperature of 110ºC in the 5th extruder zone. Under these conditions, expanded snacks were produced with 100% corn (standard) and with the incorporation of 22 and 43% of quinoa flour, and subsequently characterized, aromatized and subjected to a sensory evaluation for acceptance and purchasing intention. The expanded corn and quinoa snacks showed values for hardness and diameter similar to those available on the market, indicating that the extrusion process conditions were suitably chosen. From the nutritional point of view, the snacks with the incorporation of quinoa showed higher protein and essential amino acid (threonine, cysteine, isoleucine, lysine and tryptophan) contents and available lysine, as compared to the 100% corn snacks, demonstrating the feasibility of adding quinoa flour to corn products to increase their nutritional value. The expanded snack with 22% of quinoa flour was well accepted and showed high purchasing intention, whereas the snack with 43% was not well accepted because it showed less expansion, darkening and an aftertaste / Doutorado / Tecnologia de Alimentos / Doutor em Tecnologia de Alimentos

Orientador: Paulo Arruda / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-26T17:19:31Z (GMT). No. of bitstreams: 1 Kiyota_Eduardo_D.pdf: 12985637 bytes, checksum: 423c7614185847e8e3a4c43acbef92fe (MD5) Previous issue date: 2015 / Resumo: O amino ácido lisina é catabolizado em plantas e animais pela via da sacaropina. Nesta via, a lisina é convertida a ?-aminoadipato-?-semialdeído (AASA) pela ação da enzima bifuncional lisina-cetoglutarato redutase/sacaropina desidrogenase (LKR/SDH). O intermediário AASA é então convertido a ?-aminoadipato (AAA) pela enzima ?-aminoadipato-?-semialdeído desidrogenase (AASADH). A LKR/SDH já foi bem caracterizada em plantas e animais, mas a atividade enzimática bem como o possível papel fiiológico da AASADH ainda não foi demonstrada em plantas. A via da sacaropina, além do seu importante papel na regulação dos níveis de lisina, está também envolvida em processos de resposta a estresses. Este trabalho está dividido em dois capítulos. No capítulo I descrevemos a identificação do gene que codifica a AASADH em milho e a caracterização da atividade enzimática da enzima em endosperma imaturo de milho. Mostramos que a AASADH é codificada pelo gene Aldh7b1, um gene muito conservado em eucariotos. A enzima codificada pelo gene Aldh7b1 foi parcialmente purificada de endosperma imaturo de milho e através de eletroforese em condições denaturantes e cromatografia em coluna de gel filtração mostramos que a enzima, na sua forma nativa, apresenta-se como um tetrâmero constituído por quatro subunidades de 55 kDa. A AASADH isolada de endosperma imaturo de milho converte o semi-aldeido AASA em AAA. O produto da reação catalisada pela AASADH foi confirmado por cromatografia em camada delgada. No capítulo II discutimos o papel da via sacaropina no desenvolvimento da semente e na resposta de planas jovens de milho a estresses abióticos. As enzimas LKR/SDH e AASADH são co-expressos nas células das camadas da sub-aleurona do endosperma de milho nas fases intermediarias do desenvolvimento. No entanto, embora a proteína AASADH seja produzida no endosperma e no embrião de sementes imaturas e nos tecidos de plantas jovens, a proteína LKR/SDH é detectada unicamente nas células da sub-aleurona das sementes imaturas. A AASADH mostrou atividade máxima a pH 7,4 e Kms para AASA e NAD+ na ordem de micromolar. Em endosperma imaturo a via da sacaropina é induzida por lisina e reprimida por estresse salino, enquanto prolina e ácido pipecólico são significativamente reprimidos por lisina. Em coleóptiles jovens as enzimas LKR/SDH e AASADH são induzidas transcricionalmente por estresses salino, osmótico e oxidativo, mas enquanto que a proteína AASADH acumula nos tecidos sob estresse, a proteína LKR/SDH não é detectada. Nossos resultados indicam que os genes que codificam as enzimas LKR/SDH e AASADH são co-expressos a nível transcricional, mas não a nível traducional. A ausência da proteína LKR/SDH em plantas jovens sob estresses e os altos níveis do seu transcrito serem detectados mostra um desacoplamento transcrição/tradução que podem ter consequências regulatórias ainda desconhecidas / Abstract: Lysine is catabolized in developing plant tissues through the saccharopine pathway. In this pathway, lysine is converted into ?-aminoadipate-?-semialdehyde (AASA) by the bifunctional enzyme lysine-ketoglutarate reductase/saccharopine dehydrogenase (LKR/SDH). AASA is then converted into ?-aminoadipate (AAA) by aminoadipic semialdehyde dehydrogenase (AASADH). LKR/SDH was characterized in higher eukaryotes, but AASADH has not been demonstrated in plants. Furthermore, studies have shown that besides the degradation of lysine, the saccharopine pathway is involved in stress response processes in plants, animals and bacteria. This work was divided into two chapters. Chapter I describes the identification of the gene encoding AASADH and the partial purification and characterization of the enzyme from developing maize endosperm. The enzyme AASADH is encoded by the Aldh7b1 gene, a gene highly conserved among eukaryotes. The enzyme partially purified from developing endosperm and analyzed by SDS-PAGE and gel filtration chromatography behaved, in its native form, as a tetramer constituted by four monomers of 55 kDa. The enzymatic convertion of AASA into AAA was verified by thin layer chromatography. In Chapter II the role of the saccharopine pathway in seed development and stress responses is discussed. LKR/SDH and AASADH are co-expressed in the sub-aleurone cell layers of the developing endosperm; however, although AASADH protein is produced in reproductive and vegetative tissues, the LKR/SDH protein is detectable only in the developing seeds. AASADH showed an optimum pH of 7.4 and Kms for AASA and NAD+ in the micromolar range. In the developing endosperm the saccharopine pathway is induced by exogenous lysine and repressed by salt stress, whereas proline and pipecolic acid synthesis are significantly repressed by lysine. In young coleoptiles the LKR/SDH and AASADH transcriptions are induced by abiotic stress, but while the AASADH protein accumulates in stressed tissues, LKR/SDH does not. Our results indicate that the genes encoding the LKR/SDH and AASADH enzymes are co-expressed at the transcriptional level, but not the translational level. The absence of LKR/SDH protein in young plants under stress despite of the high levels of transcripts being detected suggests a decoupling transcription/translation that may have regulatory consequences yet unknown / Doutorado / Genetica Vegetal e Melhoramento / Doutor em Genetica e Biologia Molecular

Biacetilo (2,3-butanediona) é um contaminante de comida e cigarro, também implicado na hepatoxicidade do álcool e em doenças pulmonares. O metilglioxal (MG), um &#945;-oxoaldeído reativo frequentemente associado ao diabetes e envelhecimento, é produto da fragmentação oxidativa de trioses fosfato, acetona e aminoacetona. Por sua vez, peroxinitrito - um potente oxidante, agente nitrante e nucleófilo formado in vivo pela reação controlada por difusão do ânion radical superóxido com o radical óxido nítrico (k ~1010 M-1s-1) é capaz de se adicionar a CO2 e compostos carbonílicos, gerando produtos potencialmente tóxicos ou sinalizadores celulares. Aminoácidos, peptídeos e nucleobases podem ser acetilados nos grupos amina e na porção desoxiribose. Relativamente ao tratamento com peroxinitrito isolado, níveis superiores de 3-nitrotirosina foram detectados quando tirosina foi tratada com peroxinitrito/biacetilo ou metilglioxal. Ambos os grupos amina de lisina (Lys) ou um deles de derivados de lisina bloqueados e um deles (Ac-Lys-OMe, Z-Lys-OMe) foram acetilados pelo sistema biacetilo ou metilglioxal/peroxinitrito. Em tetrapeptídeos sintéticos contendo lisina como aminoácido amino-terminal (H-KALA-OH, Ac-KALA-OH and H-K(Boc)ALA-OH), a lisina foi acetilada pelo sistemas dicarbonilico/peroxinitrito no grupo &#945;-amina (em maior extensão) e/ou no &#949;-amina (em menor extensão). No conjunto, estes resultados podem ser interpretados à luz do mecanismo proposto para a reação de compostos &#945;-dicarbonílicos com peroxinitrito, o qual envolve sequencialmente: (i) adição nucleofílica de peroxinitrito à carbonila; (ii) homólise do aduto peroxinitroso formado, liberando &#8226;NO2 e um radical oxila do reagente carbonílico; (iii) &#946;-clivagem do radical oxila a um ácido carboxílico (ácido acético no caso de biacetilo e ácido fórmico, a partir de metilglioxal) e radical acetila; (iv) captação do radical acetila pelo oxigênio molecular dissolvido dando acetato, ou por aminoácido ou nucleobase, se presentes, gerando o produto acetilado. Tais resultados são interessantes ao levantar a hipótese de acetilação radicalar como mecanismo de modificação pós-traducional de proteínas, até então considerado um processo realizado apenas por acetilases. / Diacetyl (2,3-butanedione) is a food and cigarette contaminant recently implicated in alcohol hepatotoxicity and lung disease. In turn, methylglyoxal (MG) is an &#945;-oxoaldehyde frequently associated with diabetes and aging that is putatively formed by the oxidative fragmentation of trioses phosphate, acetone and aminoacetone. Peroxynitrite - a potent oxidant, nitrating agent and nucleophile formed in vivo by the diffusion-controlled reaction of superoxide radical with nitric oxide (k ~1010 M-1s-1) - is able to form adducts with carbon dioxide and carbonyl compounds. When initially present in the reaction mixtures before addition of ONOO-, amino acids, peptides and nucleobases undergo acetylation at the amino group and purine moieties in the presence of biacetyl or methylglyoxal. Higher levels of 3-nitrotyrosine nitration were measured when peroxynitrite/biacetyl or metilglioxal was added to tyrosine, in comparison with peroxynitrite alone. Both amino groups of L-lysine or one of the amino groups of L-lysine derivatives (Z-Lys-OH and Ac-Lys-OH) were acetylated by biacetyl and methylglyoxal/peroxynitrite system. Using tetrapeptides containing lysine at the terminal amino acid (H-KALA-OH, Ac-KALA-OH and H-K(Boc)ALA-OH), the lysine residue was acetylated at both or either &#945;-amino (major adduct) and &#949;-amino group (minor adduct). Altogether these data can be interpreted by the mechanism proposed to describe the reaction of &#945;-dicarbonyls with peroxynitrite as follows: (i) nucleophilic addition of peroxynitrite to the carbonyl group of the reagent; (ii) homolysis of the formed peroxynitroso carbonyl adduct to &#8226;NO2 and a carbonyloxyl radical; (iii) &#946;-cleavage of the oxyl radical to acetyl radical plus acetic acid (from diacetyl) or formic acid (from methylglyoxal); (iv) competitive scavenging of the acetyl radical by dissolved molecular oxygen and by added amino acid, peptide or nucleobase, ultimately yielding acetate or acetylated biomolecule. If occurring in vivo, these radical reactions may contribute to the post-translational modification of proteins catalyzed by transacetylases.

O objetivo neste trabalho foi avaliar a concentração de aminoácidos essenciais (Lisina e Metionina) considerada na literatura como ideal, de acordo com a via de fornecimento (sucedâneo ou concentrado inicial), e sua associação com a suplementação de Glutamato e Glutamina, para bezerros em dois sistemas de aleitamento: convencional ou programado. No primeiro experimento foi realizado o levantamento da composição bromatológica e em aminoácidos dos principais sucedâneos comercializados no Brasil. Nos segundo e terceiro experimentos foram utilizados 45 bezerros da raça Holandês, em delineamento de blocos casualizados distribuídos nos tratamentos: 1) Controle: sem suplementação; 2) Suplementação de lisina e metionina para atingir consumo de 17 e 5,3 g/d, respectivamente, com correção feita com base na análise do sucedâneo, 3) Suplementação de lisina e metionina, para atingir consumo de 17 e 5,3 g/d, respectivamente, com correção feita com base na análise do concentrado inicial. A diferença entre os experimentos foi o sistema de aleitamento ao qual os bezerros foram submetidos: no segundo experimento os animais receberam 6L/d de sucedâneo; enquanto no terceiro estudo os animais foram submetidos ao sistema de aleitamento programado (4L/d até a 2ª semana; 8L/d da 3ª a 6ª semana; 4L/d da 7ª a 8ª semana). No quarto experimento o mesmo delineamento foi utilizado para avaliar, em sistema de aleitamento convencional, os tratamentos: 1) Controle: sem suplementação; 2) Sucedâneo lácteo suplementado com Lisina e Metionina, para atingir consumo de 17 e 5,3g/d, respectivamente + 1% na MS de produto contendo 10% de glutamato e de glutamina; 3) Sucedâneo lácteo suplementado com Lisina e Metionina para atingir consumo de 17 e 5,3g/d + 0,6% na MS de produto contendo 10% de glutamato e de glutamina. Os animais foram alojados em abrigos individuais, com livre acesso a água e concentrado inicial. O consumo de concentrado inicial e o escore fecal dos animais foram registrados diariamente. Semanalmente, foram realizadas pesagens e medidas de crescimento corporal. Nas semanas 2, 4, 6, 8 e 10 de vida foram realizadas colheitas de sangue para determinação de metabólitos como marcadores do status protéico dos animais (albumina, proteína total, N-ureico), status energético (glicose e ?-hidroxibutirato), crescimento ósseo (fosfatase alcalina) e crescimento muscular (creatinina). A composição em aminoácidos dos sucedâneos comercializados no Brasil foi menor que o esperado para dieta que substitui o leite integral. Nos experimentos 2 e 3 a suplementação com lisina e metionina no sucedâneo ou no concentrado inicial para bezerros não resultou em benefícios no desempenho ou no metabolismo. No estudo 4, a suplementação do sucedâneo com lisina, metionina em associação com glutamato e glutamina não alterou o desempenho, a saúde intestinal ou o metabolismo de bezerros leiteiros. / The aim in this work was to evaluate the concentration of essential amino acids (Lysine and Methionine) considered in the literature as ideal, according to feeding route (milk replacer or starter concentrate), and its association with the supplementation of glutamate and glutamine to calves in two feeding systems: conventional or step-down. In the first study, the chemical composition was analyzed and in amino acids of main milk replacer marketed in Brazil. In the second and third studies, 45 Holstein calves were used, in randomized blocks distributed in treatments: 1) Control: without supplementation; 2) Supplementation with lysine and methionine to reach consumption of 17 and 5.3 g/d, respectively, with correction based on the analysis basis of the milk replacer, 3) Supplementation of lysine and methionine to reach consumption of 17 and 5.3 g/d, respectively, with correction based on the analysis basis of starter concentrate. The difference between the experiments was the feeding system applied to the calves: in the second study, the animals received 6 L/d of milk replacer; while in the third study, the animals were submitted to the step-down system (4L/d until the 2nd week; 8L/d of the 3nd to 6th week; 4L/d of the 7th to 8th week). In the fourth study, the same experimental design was used to evaluate, in a conventional feeding system, treatments: 1) Control: without supplementation; 2) AminoGut 0.6%: milk replacer supplemented with Lysine and Methionine, to reach consumption 17 and 5.3g/d, respectively + 0.6% product containing 10% of glutamate and glutamine; 3) AminoGut 1%: milk replacer supplemented with Lysine and Methionine to reach consumption 17 and 5.3g/d + 0.6% product containing 10% of glutamate and glutamine. The animals were housed in individual hutches, with free access to water and starter concentrate. The consumption of starter concentrate and fecal scores of animals were monitored daily. Body growth was weighed and measured weekly. In weeks 2, 4, 6, 8 and 10, blood samples were collected to determine the metabolites as markers of protein status of animals (albumin, total protein, N-urea), energy status (glucose and BHBA), bone growth (alkaline phosphatase) and muscular growth (creatinine). The composition of amino acids of the milk replacer marketed in Brazil was lower than expected for diet that replaces the whole milk. In study 2 and 3, the supplementation of the milk replacer or starter concentrate with lysine and methionine resulted in no benefit on dairy calves performance or metabolism. In study 4 the supplementation of the milk replacer with lysine and methionine in association with glutamate and glutmine had no effect on performance, gut health nor metabolism of dairy calves.

Rumen protected Methionine (MET) and Lysine (LYS) are critical for milk protein synthesis in dairy cows. N-acetyl-L-methionine (NALM) is a MET derivative that consists of L-Met protected with an acetyl group that is attached to the α-amino group.N-acetyl-L-lysine (NALL) is a LYS derivative that is similarly protected. The objectives of these studies were to quantify the gastrointestinal availability of NALM and NALL. Three experiments were run as 3 × 3 Latin square using 3 second lactation Holstein cows that have been fitted with cannulas in the rumen and duodenum. The cows were fed diets containing the supplements for two weeks prior to each experiment so that the rumen microbes had time to adjust to the supplement. Each period consisted of 10 d of adaptation followed by 2 d of sampling. A dose of 0, 30, or 60 g of NALM was placed under the rumen mat at the time of feeding every day during experiment 1. The cows were similarly supplied with 0, 60, or 120 g of ƐNALL during experiment 2. The cows were supplemented with 0 g, 120 g ofƐNALL, or 120 g of diNALL during experiment 3. On day one of sampling, a liquid marker (Co-EDTA) was also administered at the time of the protected AA administration. Blood, ruminal, and duodenal samples were taken at hours 0, 1, 3, 6, 9, 12, and 24 post-feeding. There were no differences for milk production, milk protein, milk fat, or DMI for NALM or either NALL. There were no differences for ruminal escape (69.1% and 46.2% respectively) and duodenal appearance (2.16% and 3.40% respectively). The ruminal escape of ƐNALL was not different between the 120 g dose (32.7%) and the 60 g dose (27.2%). Duodenal appearance was higher (P < 0.01) for the 60 g dose (2.86%) than for the 120 g dose (1.19%) of ƐNALL. Acetate, propionate, butyrate, and valerate were higher (P < 0.01) for the supplemented cows during experiment 1 with NALL. There were no differences between ƐNALL and diNALL for rumen escape, duodenal appearance, VFA production, or blood LYS AUC. Results of the experiment verify significant protection of the N-acetyl MET and LYS from rumen degradation.

Hollow fiber membranes (HFMs) formed through phase inversion methods exhibit specific physicochemical characteristics and generally favorable surface and mechanical properties, supporting their use in diverse applications including ultrafiltration, dialysis, cell culture, bioreactors, and tissue engineering. Characterization of, and modifications to, such membranes are important steps in achieving desired characteristics for specific applications. HFMs subject to gas, irradiation, and chemical sterilization techniques were characterized based on several analytical techniques. It was revealed that these common sterilization techniques can cause inadvertent changes to HFM properties. While these changes may cause detrimental effects to HFMs used in filtration, the methods of sterilization are also presented as a facile means of tuning properties toward specific applications. Modifications to HFM surface chemistries were also sought as a method of adsorbing bacterial lipopolysaccharide (LPS) from solutions used in hemodialysis treatments and bioprocessing applications. It was found that additives such as polyvinylpyrrolidone (PVP), polyethyleneglycol (PEG), and poly-L-lysine (PLL) can facilitate adsorption capacities of HFMs toward LPS. Additionally, chemical changes are presented as a means of preferentially adsorbing LPS to specific locations on the HFM surface.

Indiana University-Purdue University Indianapolis (IUPUI) / Accurate DNA replication is vital for maintaining genomic stability. Consequently, the machinery required to drive this process is designed to ensure the meticulous maintenance of information. However, random misincorporation of errors reduce the fidelity of the DNA and lead to pre-mature aging and age-related disorders such as cancer and neurodegenerative diseases. Some of the incorporated errors are the result of the error prone DNA polymerase alpha (Pol α), which initiates synthesis on both the leading and lagging strand. Lagging strand synthesis acquires an increased number of polymerase α tracks because of the number of Okazaki fragments synthesized per round of the cell cycle (~50 million in mammalian cells). The accumulation of these errors invariably reduces the fidelity of the genome. Previous work has shown that these pol α tracks can be removed by two redundant pathways referred to as the short and long flap pathway. The long flap pathway utilizes a complex network of proteins to remove more of the misincorporated nucleotides than the short flap pathway which mediates the removal of shorter flaps. Lysine acetylation has been reported to modulate the function of the nucleases implicated in flap processing. The cleavage activity of the long flap pathway nuclease, Dna2, is stimulated by lysine acetylation while conversely lysine acetylation of the short flap pathway nuclease, FEN1, inhibits its activity. The major protein players implicated during Okazaki fragment processing (OFP) are known, however, the choice of the processing pathway and its regulation by lysine acetylation of its main players is yet unknown. This dissertation identifies three main findings: 1) Saccharomyces cerevisiae helicase, petite integration frequency (Pif1) is lysine acetylated by Esa1 and deacetylated by Rpd3 regulating its viability and biochemical properties including helicase, binding and ATPase activity ii) the single stranded DNA binding protein, human replication protein A (RPA) is modified by p300 and this modification stimulates its primary binding function and iii) lysine acetylated human RPA directs OFP towards the long flap pathway even for a subset of short flaps.

Dietary inclusion of sufficient lysine is very critical for optimizing pig’s growth performance. The objectives of this project were to study the effects of dietary lysine at different concentrations on (1) the growth performance and carcass characteristics, (2) the muscle gene expression profile and the possible alterations to the metabolic and signaling pathways, (3) the muscle fatty acid profile, and (4) the plasma concentrations of growth-related hormones of late-stage finishing pigs. Nine crossbred barrows were assigned to 3 dietary treatments (lysine-deficient, equate, and -excess diets) according to a completely randomized experimental design. During the 5-week feeding trial, pigs were allowed ad libitum access to experimental diets and water. All pigs and experimental diets were weighed individually each week during feeding trial to determine growth performance. After harvest, the carcass characteristics were determined and muscle samples were collected from longissimus dorsi for mRNA and fatty acid profiling, while the jugular vein blood was collected at the end of four weeks for analyses of three growth-related hormones. While the average daily gain showed a quadratic relationship, the dressing percentage and total lean cut weight both increased linearly with dietary lysine concentrations. Results of muscle gene expression data showed that dietary lysine deficiency may lead to decreased protein synthesis, increased protein degradation and lipid accumulation, while dietary lysine excess may lead to decreased protein degradation and increased lipid biosynthesis. Fatty acid (FA) composition data showed that different dietary lysine concentrations altered the intramuscular fat (IMF) content and FA composition, especially the unsaturated FAs. In particular, dietary lysine deficiency increased the IMF content and the proportion of mono-unsaturated FAs. Hormone analyses showed that the plasma concentrations of insulin and growth hormone were not affected by dietary lysine, whereas the concentration of insulin-like growth factor 1 was decreased by either dietary lysine deficiency or excess. Collectively, lysine may function as a signaling molecule to regulate the expression of genes related to protein turnover and lipid metabolism in the muscle of finishing pigs, causing differences in growth performance, carcass characteristics, and FA composition. IGF-1 may be a controlling growth factor that is sensitive to dietary lysine.

Salicylic acid is widely studied for its role in biotic stress signaling in plants. Several SA-binding proteins, including SABP2 (salicylic acid-binding protein 2) has been identified and characterized for their role in plant disease resistance. SABP2 is a 29 kDA tobacco protein that binds to salicylic acid with high affinity. It is a methylesterase enzyme that catalyzes the conversion of methyl salicylate into salicylic acid required for inducing a robust systemic acquired resistance (SAR) in plants. Methyl salicylic acid is one of the several mobile SAR signals identified in plants. SABP2-interacting protein 428 (SIP428) was identified in a yeast two-hybrid screen using tobacco SABP2 as a bait. In silico analysis shows that SIP428 possesses the SIR2 (silent information regulatory 2)-like conserved motifs. SIR2 enzymes are orthologs of sirtuin proteins that catalyze the NAD+-dependent deacetylation of Nε lysine-acetylated proteins. The recombinant SIP428 expressed in E. coli exhibits SIR2-like deacetylase activity. SIP428 shows homology to Arabidopsis AtSRT2 (67% identity), which is implicated in SA-mediated basal defenses. Immunoblot analysis using anti-acetylated lysine antibodies showed that the recombinant SIP428 is lysine acetylated. The expression of SIP428 transcripts was moderately downregulated upon infection by TMV. In the presence of SIP428, the esterase activity of SABP2 increased modestly. The interaction of SIP428 with SABP2, it's regulation upon pathogen infection, and similarity with AtSRT2 suggests that SIP428 is likely to play a role in stress signaling in plants.